Androgen Receptor (AR) is critical for the development of the sexually dimorphic phenotypes in many different mammalian tissues, including cartilage, bone, muscle, fat, skin, and secretory glands. Despite the importance of this gene in both normal development and human disease, little is currently known about the mechanisms that control where and when AR is normally expressed. The AR coding region is surrounded by over 1 MB of intergenic DNA. These flanking regions contain numerous short non-coding sequences that are highly conserved in different mammals. I have used comparative genomics to show that humans have an unusual deletion in the flanking regions of the AR gene, which removes non-coding sequences that are otherwise highly conserved in the chimpanzee, rhesus, dog, and mouse. I have used functional enhancer assays in transgenic mice to show that the homologous chimpanzee and mouse sequences drive reproducible expression in the developing facial vibrissae and genital tubercle, two anatomical locations, where humans are also morphologically distinct from most other mammals. The objectives of my research are to determine the significance of this deletion for human biology and to identify cis-regulatory mechanisms that specify the anatomical distribution of AR expression during mammalian development. I will utilize an inducible Cre-recombinase reporter assay to further characterize the expression pattern and cell lineages specified by the AR control region that is missing in humans. To rigorously test the functional consequence of the loss of this conserved sequence, I will also use a targeted gene knock-in strategy to delete this element in the mouse and replace it with that of the chimpanzee, thus replicating both the human and chimpanzee genetic conditions in this enhancer region. Finally, I will also scan a large 650 kb intergenic region surrounding the AR gene for other tissue specific and developmental enhancers, using bacterial artificial chromosome (BAC) clones and functional assays in transgenic mice. These experiments will greatly improve our understanding of the mechanisms by which AR expression is established during mammalian development and sexual differentiation, and the role it plays in specifying the unique human phenotype. This research is relevant to public health because the misexpression of AR is associated with multiple human diseases such as androgen insensitivity syndrome, spinal bulbar muscular atrophy, and prostate cancer. Identifying the mechanisms by which AR expression is regulated may aid in the long-term diagnosis and treatment of these and other human diseases.